Project Summary/Abstract While acute myeloid leukemia (AML) is the most common form of leukemia in adults, prognosis remains poor. An emerging strategy for the treatment of AML centers around inhibiting the serine proteases DPP8 and DPP9 (DPP8/9), which activate the CARD8 inflammasome and triggers a lytic form of cell death known as pyroptosis. This process is directly cytotoxic to AML cells and indirectly activates the host immune response, delivering a dual mechanism for anticancer activity. Our central hypothesis is that DPP8/9 processing inactivates some as of yet unknown substrate(s) that, when active, recruit the inflammasome and triggers pyroptosis. Unfortunately, DPP8/9, like many of the ~550 human proteases, remain uncharacterized with respect their endogenous substrates. Identifying and characterizing the physiologically relevant substrates of DPP8/9 will be necessary to both understand and therapeutically exploit this pyroptotic checkpoint. Although proteases regulate countless important (patho)physiological processes, discovering protease substrates is extraordinarily challenging and state-of-the-art protease substrate profiling platforms are expensive and incomprehensive. In Specific Aim 1, I will advance a practical technology for protease substrate identification. This innovative platform uses an N-terminal reactive chemical moiety to selectively label the N-termini of proteins in complex cell lysates. Coupling this chemical group to biotin will enable the labeling, enrichment, and identification of endogenous protease substrates. In Specific Aim 2, I will use this technology, along with complimentary strategies to identify the substrates of DPP8/9, and then characterize how these substrates activate the CARD8 inflammasome. I anticipate that this project will lead to two key outcomes: 1) the introduction of a practical method for protease substrate discovery that will enable the unbiased interrogation of the many complex proteolytic networks that control important biological responses, and 2) the characterization of the DPP8/9 ? CARD8 pyroptotic checkpoint, which has high potential to enable modulation of this pyroptotic pathway for the treatment of AML.